We are using peptide mass mapping and MALDI-PSD-MS to determine the amino acid sequences of the protein osteocalcin in bones of ancient organisms and their closest modern ancestors to establish preliminary evolutionary relationships. The use of osteocalcin and the application of MALDI-MS provide a unique combination for the project. Osteocalcin has several important attributes which include intimate association with the hydroxyapatite mineral phase of bone, preferential preservation over long time periods, exclusive occurrence in vertebrate mineralized tissues, absence in common contaminators including microbes, invertebrates and plants, retention of immunological specificity via radioimmunoassay (RIA), and the existence of an extensive database of protein sequences for osteocalcin. In our particular case, prior work has confirmed the presence of osteocalcin via RIA for all ancient samples. The MALDI-MS methods are not subject to the myriad of problems that confront traditional methods for protein sequencing. Although this approach has never been attempted on ancient proteins, we intend to demonstrate its potential to delineate ancient protein sequences. Given the exclusive yet ubiquitous occurrence of osteocalcin among vertebrates, the techniques developed in this project will be directly applicable for molecular studies on any number of other vertebrate lineages with obscured evolutionary relationships.